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Evasion and Immuno-Endocrine Regulation in Parasite Infection: Two Sides of the Same Coin in Chagas Disease?

1Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

2Institute of Clinical and Experimental Immunology of Rosario, CONICET, National University of Rosario, Rosario, Argentina

3Faculty of Medical Sciences, National University of Rosario, Rosario, Argentina

Chagas disease is a serious illness caused by the protozoan parasite Trypanosoma cruzi. Nearly 30% of chronically infected people develop cardiac, digestive, or mixed alterations, suggesting a broad range of host-parasite interactions that finally impact upon chronic disease outcome. The ability of T. cruzi to persist and cause pathology seems to depend on diverse factors like T. cruzi strains, the infective load and the route of infection, presence of virulence factors, the parasite capacity to avoid protective immune response, the strength and type of host defense mechanisms and the genetic background of the host. The host-parasite interaction is subject to a constant neuro-endocrine regulation that is thought to influence the adaptive immune system, and as the infection proceeds it can lead to a broad range of outcomes, ranging from pathogen elimination to its continued persistence in the host. In this context, T. cruzi evasion strategies and host defense mechanisms can be envisioned as two sides of the same coin, influencing parasite persistence and different outcomes observed in Chagas disease. Understanding how T. cruzi evade host's innate and adaptive immune response will provide important clues to better dissect mechanisms underlying the pathophysiology of Chagas disease.

Introduction

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (WHO, 2015). Nearly 30% of chronically infected people develop cardiomyopathy, megacolon, and megaesophagus or a mixed of these alterations, suggesting a broad range of host-parasite interactions that finally impact upon chronic disease outcome (Rassi et al., 2010). Different and not mutually exclusive hypotheses have been considered for the pathogenesis of chronic Chagas disease, including autoimmunity by molecular mimicry, microvascular (Ramos and Rossi, 1999), and autonomic dysfunction (Dávila et al., 2004), and tissue damage by parasite persistence (Gironès et al., 2005; Gutierrez et al., 2009; Cunha-Neto et al., 2011). The parasite persistence hypothesis predicts a chronic inflammatory reactivity as result of a failure in parasite burden control, thus promoting the development of disease pathology (Tarleton, 2001). In addition, a subpatent parasite-induced cell lysis as consequence of amastigote differentiation into trypomastigotes (Bonney and Engman, 2008) might fuel inflammation. The presence of parasites (Añez et al., 1999; Buckner et al., 1999) or their products, such as DNA, in blood and myocardium of chronic infected hosts is well documented (Añez et al., 1999; Zhang and Tarleton, 1999; Salomone et al., 2000; Elias et al., 2003). T. cruzi reactivation in HIV co-infected, transplanted or immunocompromised chronic chagasic patients provides convincing evidence of parasite persistence (Tarleton, 2001; Andrade et al., 2014), reinforcing the view that disease pathology and its severity are directly related to T. cruzi presence within the affected tissue (Tarleton, 2001). In this review, we examined the complexity of cellular, molecular and physiologic factors involved in T. cruzi evasion and persistence in the light of current data.

Figure 1. Pathogenesis of Trypanosoma cruzi infection. Several hypotheses have been considered for the pathogenesis of chronic Chagas disease, comprising tissue damage by parasite persistence, autoimmunity, microvascular injury, and autonomic dysfunction. Since diverse factors are involved in parasite evasion and persistence, most of all may influence the infection outcome and the development of pathology in almost 30% of infected individuals. The ability of T. cruzi to evade immune system seems to depend on diverse factors like T. cruzi strains, the infective load and the route of infection and the presence of virulence factors; but also can be determined by the type and strength of host defense mechanisms and the genetic background of the host.

FIGURE 2

Figure 2. Aspects of Trypanosoma cruzi evasion and persistence in the vertebrate host. T. cruzi parasites develop different strategies to evade the host defenses and establish a persistent infection. T. cruzi parasites evade the host innate immune responses associated with macrophage and complement system (A). The trans-sialidase (TS), a T. cruzi-derived virulence factor, can also overcome the host resistance responses to optimize the invasion and parasite persistence in chronic infection (B). The development of anti-parasite immune response is coupled with the activation of neuroendocrine axes that may affect the course of disease (C). Adipose tissue can be considered as a parasite reservoir and may contribute to the establishment of persistent infections, playing a major role in T. cruzi burst during immunosuppression periods (D). The recognition of T. cruzi-derived antigens in the thymus may restrict the central tolerance to parasite infection, while the release of immature and potentially autoimmune T cells to the peripheral non-lymphoid tissues may be related with disease pathology in the chronic phase (E).

Adipose Tissue Acts as a Reservoir of Trypanosoma cruzi

Another adaptive strategy displayed by T. cruzi parasites to optimize its persistence in the host consists in targeting tissues with particular characteristics. Some studies have shown that adipose tissue (AT) might serve as a reservoir where parasite could persist in a latent state to avoid the host-defense mechanisms, acting as a possible site of reactivation, similarly to that observed for other intracellular pathogens (Figure 2B; Neyrolles et al., 2006; Bechah et al., 2010, 2014). Adipocytes could harbor a significant number of parasites even in the chronic phase of infection (Combs et al., 2005). Recently, more robust evidence that AT can act as a reservoir of T. cruzi have arisen from experiments in which infected mice were subsequently treated with an anti-parasitic drug and afterwards immunosuppressed. Intriguingly, in a significant number of animals, the AT was the major site of parasite recrudescence (Fortes Francisco et al., 2015). Moreover, studies carried out in patients with chronic chagasic cardiomyopathy have revealed the presence of parasite-derived DNA in AT (Ferreira et al., 2011). In this sense, AT may be a reservoir from which reactivation of infection may occur, especially during periods of immunosuppression, as observed in chagasic patients co-infected with HIV, transplanted or undergoing immunosuppressive therapies (Almeida et al., 1996; Sartori et al., 1998; Campos et al., 2008; Cordova et al., 2008; Pinazo et al., 2013). Moreover, in HIV co-infected chagasic patients, periods of lipoatrophy may result in the release of parasites into the circulation (Ferreira et al., 2011). It remains to be investigated why T. cruzi persists in the AT. Possible reasons could be the special metabolic conditions that T. cruzi finds inside the adipocyte and the slow turnover of these cells. After infection, there is an influx of inflammatory cells to AT, accompanied by an in situ upregulation of both TNF-α and IL-6, concomitantly to a diminution of adipocytokine levels (Desjardins and Descoteaux, 1997; Chandrasekar et al., 2000; Manarin et al., 2013). Moreover, some studies suggested that metabolic alterations induced by T. cruzi persistence in AT may increase the risk of diabetes, metabolic syndrome and cardiovascular disease (Chandrasekar et al., 2000; Nagajyothi et al., 2009; Manarin et al., 2013).

Recently, it has been shown that in vivo exposure to T. cruzi mucins enhances the host susceptibility, as seen by the increased parasitemia and heart tissue damage. These effects were associated with a reduction in Th1 and Th2 cytokine production, together with decreased levels in the frequency of IFN-γ producing CD4+ T cells in the spleen of mice treated with parasite mucins in comparison with untreated controls (Nunes et al., 2013). Interestingly, it has been shown that the binding of acid-binding Ig-like lectin Siglec-E (CD33) by T. cruzi mucins inhibits the mitogenic responses of CD4+ T cells. Studies conducted to address the molecular mechanisms underlying these effects have shown that the impairment of TCR/CD3-mediated activation of CD4+ T cells by T. cruzi-derived mucins was correlated with induction of G1-phase cell cycle arrest. Importantly, it has been demonstrated that interactions of the terminal sialyl residues of T. cruzi mucins with CD4+ T cells led to the induction of the cell cycle regulator p27/Kip1 responsible to block the transition from G1 to S phase of mytosis, thus preventing the proliferative responses (Nunes et al., 2013).

Interestingly, the limited T cell responses observed in T. cruzi infection contrast with the large polyclonal expansion of B lymphocytes seen in the acute phase (Ortiz-Ortiz et al., 1980), as demonstrated by the increased frequency of IgG2a and IgG2b secreting B cells in peripheral lymphoid organs of infected mice. This phenomenon results in high frequency of nonspecific antibodies with low affinity for T. cruzi antigens (Ouaissi et al., 2001), some of them cross-reacting with heart and neural autoantigens (Acosta and Santos-Buch, 1985; Kierszenbaum, 1999; Engman and Leon, 2002). The auto-reactive B cell responses are thought to play secondary roles in the pathogenesis of Chagas disease. The extensive polyclonal expansion of the B cells could partly affect lymphoid compartments by increasing the competition for activation and survival signals needed to promote the generation of antigen-specific lymphocyte responses against T. cruzi (Freitas and Rocha, 2000; Montaudouin et al., 2013).

In addition, parasite-derived glycol-inositol-phospholipids (GIPLs), which are components of the dense glycolipid layer covering the parasite cell surface, also promote alterations in the B cell compartment. These molecules work as TLR4 agonists, mediating pro-inflammatory effects (Oliveira et al., 2004). Another virulence factor encoded by T. cruzi that target the B cell compartment is the proline racemase, which participates in arginine and proline metabolism, acting as a potent mitogen for B cells. Shortly, T. cruzi -derived virulence factors are active players in the subversion of the host immune system and are determinant for the establishment of chronic persistent infection (Reina-San-Martín et al., 2000; Chamond et al., 2003).

The Immune-Endocrine Imbalance is a Key Determinant of Parasite Persistence

Immune and neuro-endocrine systems are integrated through a complex network of mediators, involving cytokines, adipocytokines, hormones, and neuropeptides that collectively act to maintain homeostasis (Besedovsky and del Rey, 1996; Fantuzzi, 2005). However, when vertebrate hosts are challenged by infectious pathogens, acute and short-term stress signals are delivered by this network to initiate and build global host mechanisms of defense (Besedovsky and del Rey, 1996). In parallel, pathogens could interfere with this neuro-endocrine response at several levels. Thus, a race between pathogen-mediated evasion mechanisms and host immune response will determine whether the microorganisms will be rapidly eliminated or persist in the host (Figure 2D). In mice, the anti-T. cruzi immune response is associated with the activation of neuro-endocrine circuitries, mainly the hypothalamic-pituitary-adrenocortical (HPA) axis (Roggero et al., 2006; Corrêa-De-Santana et al., 2006b). In this scenario, pro-inflammatory cytokines released during infection activate the HPA axis, leading to production of glucocorticoids (GC), crucial for host survival. Evidently, the neuro-endocrine circutries initiates an anti-inflammatory response attempting to minimize the infection-induced collateral tissue damage. However its immunoregulatory effect ultimately favor the parasitism and establishment of persistent infection. Comparative studies between susceptible and resistant experimental mice models have indicated that the course of T. cruzi infection strongly depends on the appropriate timing and magnitude of the immune-endocrine response (Roggero et al., 2006). Susceptible animals succumb as consequence of increased inflammatory response poorly counteracted by the HPA axis, while resistant animals develop a more balanced immune-endocrine response that lead to the establishment of a chronic infection and mild pathology. Moreover, when GC signaling was abrogated by adrenalectomy or treatment with GC receptor antagonist RU486, the severity of infection increased dramatically as a result of an augmented inflammation-based immunopathology (Roggero et al., 2006; Pérez et al., 2007). These findings indicate that a delicate balance between the immune and endocrine systems play a role in the establishment of chronic infections. Additionally, the activation of HPA axis leads to secretion of other adrenal steroids, such as dehydroepiandrosterone (DHEA). In this regard, the increased vulnerability of T. cruzi infected young animals was associated with a high corticosterone/DHEA-sulfate ratio as compared to the adult counterparts (Pérez et al., 2011). Similarly, patients with severe chronic chagasic myocarditis also revealed a disruption in the activation of HPA axis as characterized by decreased concentrations of DHEA-sulfate and unbalanced cortisol/DHEA-sulfate ratio in comparison to asymptomatic or healthy individuals (Pérez et al., 2011). Overall, these findings reinforce the view that during T. cruzi human infection there are endocrine disturbances that might favor parasite persistence, thus influencing the disease pathology.

Moreover, pro-inflammatory cytokines associated to T. cruzi infection such as TNF-α, IL-6, or IL-1β could affect the release of hypothalamic, pituitary or adrenal hormones by their direct action on the endocrine glands (Kanczkowski et al., 2013, 2015; Hueston and Deak, 2014). During experimental T. cruzi infection, TNF-α has been implicated in the HPA activation at central level (Roggero et al., 2006; Pérez et al., 2007), although inhibitory actions at adrenal level has been also observed (Villar et al., 2013). Acutely infected TNF-R1 knock-out mice showed an enhanced transcription of adrenal steroidogenic proteins StAR, CYP11A1, CYP11B1 and 11β-HSD1 as compared to wild type mice, suggesting that GC secretion can be down regulated by TNF-α in situ, independently of the signaling pathway induced by adrenocorticotropic hormone (ACTH; Corrêa-De-Santana et al., 2006a; Villar et al., 2013). Since both parasites and their antigens had been detected within adrenal glands (Corrêa-De-Santana et al., 2006a; Villar et al., 2013), their presence might induce in situ the release of TNF-α, with the consequent modulation of GC secretion. In addition, IL-6 has also been associated with enhanced activity of the HPA axis during experimental T. cruzi infection. In this regard, supernatants of adenopituitary cell cultures challenged with the parasite contained more IL-6, while infected mice also showed augmented circulating levels of this cytokine systemically (Corrêa-De-Santana et al., 2006a). The activation of hypothalamus-pituitary unit also results in the release of both growth hormone (GH) and prolactin (PRL), which are capable of improving the immune response, counteracting the GC-driven immunosuppression. T. cruzi infection appears to directly modulate the secretion of both hormones, since in vitro infection of mammosomatotrophic cell line diminished GH and PRL secretion, similarly to observed in the pituitary glands of infected mice (Corrêa-De-Santana et al., 2009). The modulation of GH and PRL secretion by diminishing the Pit-1 gene expression, a major transcription factor for both hormone genes (Corrêa-De-Santana et al., 2009). Moreover, the downregulation of these hormones during the infection might be also related to the presence of parasites or their antigens in the glands, favoring T cell and macrophage infiltration, vascular stasis along with increased depots of extracellular matrix proteins (Corrêa-De-Santana et al., 2006b, 2009). The downmodulation of GH and PRL hormones is also observed in African trypanosomiasis and may illustrates a common modulatory mechanism (Radomski et al., 1994, 1996). Moreover, there is a bulk of evidence indicating that sex steroid hormones might influence the development and course of diverse parasitic infections (Romano et al., 2015). Particularly, it has been shown that T. cruzi parasites have the capacity to metabolize steroid hormones (Vacchina et al., 2008), suggesting a possible role of this mechanism in the host-parasite interplay.

The T. cruzi infected thymus undergoing atrophy is still able to carry out negative selection, remaining important considerations in the context of host-pathogen interactions (Mendes-da-Cruz et al., 2003; Morrot et al., 2011). T. cruzi parasites also colonize the thymus (Savino et al., 1989), so their antigens may be presented to recirculating parasite-specific memory T cells migrating from the periphery to the thymic microenvironment. Alternatively, the parasite colonization of thymus could lead to the generation of T. cruzi-specific Tregs with high affinity TCR (Pacholczyk and Kern, 2008), thus promoting the host tolerance to persistent infection. Interestingly, it has been observed that chagasic patients in the indeterminate phase shown high frequencies of circulating Tregs as compared to chronic cardiac ones (De Araujo et al., 2011), suggesting a beneficial role of Tregs in suppressing the pathology associated to disease progression. In contrast, in experimental lethal models of Chagas disease with highly Th1-polarized inflammatory responses, the expansion of Tregs is clearly restricted (González et al., 2015).

Concluding Remarks

Recent studies suggest that the immuno-endocrine host response may favor T. cruzi chronic persistence. Future studies attempting to understand how T. cruzi evade host immune response or the extent by which parasite persistence might be favored by immune-neuro-endocrine regulation will provide important clues to better dissect mechanisms underlying the pathophysiology of Chagas disease.

Author Contributions

AM, FBG, SV and AP wrote the paper. All authors read and approved the final version of the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Dr. Oscar Bottasso for their advices critical reading of text. This work was supported by grants from CNPq and FAPERJ (Brazil); ANPCyT (PICT 2013-1892), CONICET (PIP 0641), and ASACTeI (Argentine). AM is recipient of fellowship from CNPq and FG is recipient of fellowship from CONICET.

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